RESUMO
Campylobacter and Helicobacter species express a 6-amino-6-deoxyfutalosine N-ribosylhydrolase (HpMTAN) proposed to function in menaquinone synthesis. BuT-DADMe-ImmA is a 36 pM transition state analogue of HpMTAN, and the crystal structure of the enzyme-inhibitor complex reveals the mechanism of inhibition. BuT-DADMe-ImmA has a MIC(90) value of <8 ng/mL for Helicobacter pylori growth but does not cause growth arrest in other common clinical pathogens, thus demonstrating potential as an H. pylori-specific antibiotic.
Assuntos
Adenina/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/enzimologia , N-Glicosil Hidrolases/antagonistas & inibidores , Pirrolidinas/química , Pirrolidinas/farmacologia , Adenina/química , Adenina/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Infecções por Helicobacter/tratamento farmacológico , Humanos , Modelos Moleculares , N-Glicosil Hidrolases/metabolismoRESUMO
A series of eight novel, highly modified mono-, di- and trisaccharide derivatives, each containing an iminoalditol moiety, has been synthesized in the quest for potential heparanase inhibitors.
Assuntos
Compostos Aza/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glucuronidase/antagonistas & inibidores , Heparitina Sulfato/síntese química , Heparitina Sulfato/farmacologia , Inibidores Enzimáticos/química , Heparitina Sulfato/análogos & derivados , Heparitina Sulfato/química , HumanosRESUMO
BACKGROUND: Mutations in the skeletal muscle ryanodine receptor gene may result in altered calcium release from sarcoplasmic reticulum stores, giving rise to malignant hyperthermia (MH). MH is a pharmacogenetic skeletal muscle disorder triggered by volatile anesthetics and depolarizing muscle relaxants. Diagnosis of MH is by in vitro contracture testing of quadriceps muscle. DNA analysis of causative mutations is limited by the large number of mutations that cosegregate with MH and the relatively few that have been biochemically characterized. METHODS: DNA sequence analysis was used to screen the skeletal muscle ryanodine receptor gene in MH-susceptible individuals. A diagnostic test using real-time polymerase chain reaction was developed to detect the mutation in individuals diagnosed as MH susceptible by in vitro contracture testing. The functional relevance of this mutation was examined in Epstein-Barr virus-immortalized B-lymphoblastoid cells. RESULTS: A novel ryanodine receptor mutation (cytosine 14997 thymine resulting in a histidine 4833 tyrosine substitution) was identified in pathology specimens from two patients with fatal MH reactions. B lymphocytes from patients with this mutation were approximately twofold more sensitive than MH-negative cells to activation with 4-chloro-m-cresol. The amount of Ca released from B lymphocytes of MH-susceptible patients was significantly greater than that released from cells of family members without this mutation. Haplotype analysis suggests that both families had a common ancestor. CONCLUSIONS: DNA analysis to detect mutations which cosegregate with MH as well as biochemical assays on cultured lymphocytes obtained from blood can serve as useful diagnostic tools for MH susceptibility and genotype-phenotype correlations.
Assuntos
Hipertermia Maligna/genética , Mutação , Polimorfismo de Nucleotídeo Único , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Adolescente , DNA/sangue , DNA/genética , DNA/isolamento & purificação , Evolução Fatal , Feminino , Parada Cardíaca , Humanos , Masculino , Hipertermia Maligna/etiologia , Meningites Bacterianas/complicações , Músculo Esquelético/fisiologia , Nova Zelândia , Linhagem , Polinésia/etnologiaRESUMO
Transition states can be predicted from an enzyme's affinity to related transition-state analogues. 5'-Methylthioadenosine nucleosidases (MTANs) are involved in bacterial quorum sensing pathways and thus are targets for antibacterial drug design. The transition-state characteristics of six MTANs are compared by analyzing dissociation constants (K(d)) with a small array of representative transition-state analogues. These inhibitors mimic early or late dissociative transition states with K(d) values in the picomolar range. Our results indicate that the K(d) ratio for mimics of early and late transition states are useful in distinguishing between these states. By this criterion, the transition states of Neisseria meningitides and Helicobacter pylori MTANs are early dissociative, whereas Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, and Klebsiella pneumoniae MTANs have late dissociative characters. This conclusion is confirmed independently by the characteristic [1'- (3)H] and [1'- (14)C] kinetic isotope effects (KIEs) of these enzymes. Large [1'- (3)H] and unity [1'- (14)C] KIEs are observed for late dissociative transition states, whereas early dissociative states showed close-to-unity [1'- (3)H] and significant [1'- (14)C] KIEs. K d values of various MTANs for individual transition-state analogues provide tentative information about transition-state structures due to varying catalytic efficiencies of enzymes. Comparing K d ratios for mimics of early and late transition states removes limitations inherent to the enzyme and provides a better predictive tool in discriminating between possible transition-state structures.